The realm of nature in which we are dwelling is inhabited by innumerable higher and lower organisms, and these organisms are maintaining their life phenomena on the basis of common molecular mechanisms such as expression of proteins by means of DNAs in response to external stimuli.
In both eukaryotic cells and prokaryotic cells, rapidly responding to the temperature, signal transducers, the presence or absence of nutrients, and the like in the breeding environment is directly related to the matter of life and death of the organism. Therefore, in regard to nutrition, energy control, propagation, and signal transduction of sensory organs such as in visual and auditory senses, living cells completes relevant signal transduction in the time ranging from a few milliseconds to minutes.
Various techniques have been developed hitherto as the means to examine such life phenomena, and some of them have already been commercialized. In recent studies conducted in a wide range of life science fields, molecular imaging within individual cells and animals has become the main trend of research for certain life phenomena (Massoud, T. F. and Gambhir, S. S. Genes & Development, 2003, 17, 545-580). In particular, research and development on bioanalysis utilizing fluorescent dye proteins such as green fluorescent protein (GFP), is becoming popular, and a technology of visualizing protein-protein interactions, or degrees of activity of proteins in a signal transduction system, particularly using fluorescence resonance energy transfer (FRET; a phenomenon in which between two fluorescent molecules, the excitation energy of one fluorescent molecule is transferred to the other fluorescent molecule to emit fluorescence), namely, so-called imaging technology is on the focus of interest. On the other hand, a molecular imaging technology utilizing lighting enzymes (LE) such as firefly luciferase (FLuc) is also attracting high attention over the world, and the research and development thereof is in intense competition.
These molecular imaging techniques may be classified by the approximate signal transduction time taken from the in vivo generation of signals to the measurement of the signals, as follows.
The molecular imaging technique requiring the longest time may be exemplified by a reporter gene assay. In this assay, the intensity, property and the like of the initial exogenous stimulators can be determined, on the basis of the intensity of luminescence or fluorescence of a reporter protein which is expressed as a result of the activation of transcription factors by exogenous stimulators, or the like, as an index. This technique requires 24 hours on the average until the amount of the reporter protein is sufficiently accumulated. This technique has been widely used in the investigation of the activity of a steroid or a chemical substance.
As a molecular imaging technique requiring the second longest time, a technique based on protein splicing was introduced (Kim, S. B., Ozawa, T., Watanabe, S., Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 11542-11547). Protein splicing means a self-catalytic splicing reaction occurring between a certain protein sequence and another protein sequence. A protein which will serve as a reporter, is bisected (N-terminal side and C-terminal side) beforehand, and the bisected fragments are respectively linked to both termini of a splicing protein. And then, the fragments may be spliced to restore a full reporter protein in response to an appropriate exogenous stimulator. The resulting recovery of luminescence or fluorescence intensities were taken as an index of the intensity of the initial external stimulator. This technique requires 2 hours at the lower limit, since sufficient time is needed for the reaction between the two fragmentized protein fragments. This technique is being effectively used for imaging the intracellular trafficking of proteins, and the like.
As a molecular imaging technique requiring the third longest time, a protein self-complementation method may be mentioned. This technique makes use of an increment of the intensity of luminescence or fluorescence generated when bisected fragments of a reporter protein are approximated to recover the activity, as the signal for analysis. For instance, in order to investigate the presence or absence of certain target protein-protein interaction, the target proteins are each linked with one of the bisected fragments of a reporter protein. Later, the reporter protein fragments are sufficiently approximated only when the two target proteins interact in response to an exogenous signal. The intensity of luminescence or fluorescence of the reconstituted reporter protein triggered by the binding between the two aimed proteins as a result is taken as an index, to evaluate the intensity of the original external signal (two-molecule-format probe: WO 2002/008766, WO 2004/104222, and Kim, S. B., Awais, M., Sato, M., Umezawa, Y., and Tao, H. Anal. Chem. 2007, 79, 1874-1880). This technique is used to evaluate the activity of a steroid or a chemical substance, and thus requires a measurement time of about 20 minutes after steroid stimulation. Recently, the inventors of the present invention have developed a method of bioimaging an intramoleculer protein-protein interaction inside a single molecule, based on the principle of self-complementation of a LE, and filed a patent application on the method (Japanese Patent Application No. 2007-005144). The feature of this technique is to integrate all the components for ligand-sensing and light emission in a single molecule, and thus enable to detect a conformation change of the ligand recognition protein induced by a ligand as one-dimensional luminescence intensity mode. Moreover, as an advanced form of the previous technique, the inventors of the present invention have further developed a technique for discriminating multiple activities of a single ligand in respectively different colors (green and red), and filed a patent application on the technique (Japanese Patent Application No. 2007-202308).
As a molecular imaging method which can be performed in a time shorter than this, there is a method based on fluorescence resonance energy transfer (FRET). In this technique, the fluorescence energy transfer occurring between two fluorescent dye proteins in proximity can be used as an index to measure the degree of the interaction between the two proteins (JP-A No. 2002-017359 and JP-A No. 2007-49943). The inventors of the present invention suggest a probe capable of visualizing the binding of cGMP with its target molecule (cGMP-binding protein) based on the FRET phenomenon (JP-A No. 2002-017359), or a probe capable of similarly visualizing IP3 using the FRET phenomenon (JP-ANo. 2007-49943). This technique can achieve fluorescence imaging of the molecular phenomena in a single cell on a real-time basis. This technique provides the fastest imaging means among those that have been developed thus far.
The probes for detecting target ligands using the FRET phenomenon (JP-ANo. 2002-017359, JP-ANO. 2007-49943, JP-A NO. 2004-325253, Awais, M.; Sato, M.; Lee, X. F.; Umezawa, Y. Angew. Chem. Int. Ed. 2006, 45, 2707-2712, and Schaufele, F.; Carbonell, X.; Guerbadot, M.; Borngraeber, S.; Chapman, M. S.; Ma, A. A.; Miner, J. N.; Diamond, M. I. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9802-9807) are excellent in that, as described above, they are capable of detecting ligands in short times, and also capable of detecting agonists and antagonists each distinctively. However, the fluorescent probes have high background fluorescence due to autofluorescence, and require a high sensitivity fluorescence microscope, a precision filtering apparatus and a trained technician to measure the energy transfer of two chromophores with high precision. Also, since excitation by short wavelength light from an external source is required, it has been very difficult to perform bioanalysis in living subjects where the absorption of short wavelength light is significant.
In addition to the above-described technologies, synthetic fluorescent dye reagents such as Fura and Indo-1, have been introduced to employ intracellular molecular imaging (Schlegel, S., Steen, M., Guse, A. H. Mol. MED. 2006, 12, S23-S23 (Suppl. 1)), but these synthetic substances have problems in that they cause cytotoxicity, accumulation in some parts of cells, and lack selectivity to target ligands.
On the other hand, bioluminescence imaging is advantageous compared with fluorescence, in that:
(1) the background emission is low;
(2) no excitation by an external light is required;
(3) the apparatus is very simple, compact, and easy to be miniaturized;
(4) the emission light has excellent tissue permeability and thus is appropriate for in vivo imaging; and the like.
For this reason, the inventors of the present invention have put an effort in developing a molecular imaging probe utilizing bioluminescence. In the case of the conventional detection method for protein-protein interaction based on protein splicing or self-complementation, which method utilizes the restorability of a partitioned reporter molecule (WO 2002/008766, WO2004/104222, and Kim, S. B., Ozawa, T., Watanabe, S., Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 11542-11547), since the respective split reporter molecules are introduced into the cells as separate probe components (two-molecule-format probe), there occurs an improper stoichiometry because the respective expression levels of the probe components differ from each other. It was also strongly feared that such stoichiometric variance causes inefficiency of the probe in sensing a ligand.
Therefore, the inventors of the present invention recently developed a single-molecule-format luminescent probe comprising excellent analytical performance, in which all of the elements necessary for the ligand sensing and light emission are integrated in a single molecule (Japanese Patent Application Nos. 2007-202308 and 2007-005144). However, the probe takes approximately 10 minutes to 20 minutes from the recognition of a ligand to the light emission. The large molecular weight (92 kD) of the conventional probes imposes a burden on the cells, and thus is not adequate to trace the molecular phenomena which take short time, in the living cells.
That is, in the case of conventional bioluminescence imaging probes, no matter whether a single-molecule-format or a two-molecule-format, the molecular weights of the probes are ever heavy because a ligand recognition domain inherent to a ligand-specific receptor is simply incorporated into the probe, and luminescent enzymes with a heavy molecular weight have been used. Furthermore, the target ligand must permeate the cell membrane and reach the probe in the cytoplasm, but in the case of insulin, various growth hormones, cytokines and the like, since these molecules originally cannot permeate the cell membrane, they cannot be detected with conventional probes expressed within the cells. Even in the case of ligands which are capable of permeating the cell membrane, since the step of membrane permeation is the rate-limiting step, thus requiring a measurement time of about several ten minutes, the real time observation of various life phenomena in a cell cannot be made by any conventional means.
In order to observe life phenomena in a living cell on a real-time basis, it is most effective to carry out molecular imaging of the signal transduction of second messengers inside the living cell, and a large number of analytic technologies have been developed and proposed in relation to the molecular imaging of second messengers (particularly, calcium ions) (JP-A No. 2007-49943 and Miyawaki A., Llopsis J., Heim R., McCaffery J. M., Adams J. A., Ikura M., Tsien R. Y. Nature, 1997, 388 (6645), 882-887).
In particular, a calcium sensor emitting fluorescence based on the above-discussed principle of FRET, and includes a protein containing calmodulin, which is a calcium recognition protein, and a peptide capable of binding to calcium ion-bound calmodulin (M13), genetic mutants of GFP such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) linked to the two termini of calmodulin, as well as probes ameliorated therefrom are widely used as probes which can detect calcium ions serving as a second messenger in living cells, on a real-time basis. However, fluorescent proteins also have problems that the proteins are generally sensitively to pH, show high background fluorescence due to autofluorescence, and have larger molecular weights because two fluorescent proteins are linked to both termini of the target protein. Moreover, there is also a problem in terms of use, such as that a large instrumentation is required to detect fluorescence (JP-A No. 2007-49943).
A fluorescent molecular probe for detecting a second messenger, which probe utilizes bisected fragments of a single GFP molecule, has also been developed (Ozawa, T., Natori, Y., Sato, M., Umezawa, Y. Nat. Methods, 2007, 4(5): 413-419). However, the probe has the same intrinsic limitations of general fluorescent probes such as that due to the poor reversibility, when bisected GFP fragments are re-bound, the probe becomes inadequate for repeated use, and that GFP requires a long time (about 6 hours) for protein folding, and is prone to undergo misfolding.